Awardees

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, with a mere 13% average 5-year survival rate. Conventional chemotherapy and immunotherapy regimens have limited effectiveness, and surgical resection of the tumor is not viable for the majority of PDAC patients diagnosed with advanced metastatic disease. Our previous research has shown therapeutic induction of cellular senescence with RAS pathway targeting therapies can induce cytokine and chemokine production through the senescence-associated secretory phenotype (SASP) that can effectively activate anti-tumor T cell immunity and responses to anti-PD-1 immune checkpoint blockade. To build on these findings and overcome limitations associated with senescence-inducing therapy toxicity, here we leveraged mRNA technology to deliver specific SASP cytokines and chemokines we have found to stimulate immune responses into the suppressive PDAC TME as an immune oncology platform. We created an in vitro transcription pipeline for mRNA production and have successfully demonstrated delivery of multiple mRNAs simultaneously into the TME of transplanted and autochthonous PDAC mouse models, stimulating the local production of cytokines normally absent in PDAC. We further characterized a novel combination of five cytokines and chemokines that can effectively recruit and activate both innate and adaptive immune responses against PDAC. Considering the recent success of off-the-shelf neoantigen mRNA vaccines in early human PDAC patient trials, we have now developed mRNAs that encode PDAC-specific antigens. By integrating our cytokine mRNA platform with antigen mRNAs, we observe a significant increase in intratumoral antigen-presenting dendritic cell populations, leading to substantially enhanced T-cell-mediated anti-tumor immune response. Long-term studies in autochthonous PDAC mouse models demonstrate that combining cytokine and antigen mRNAs can completely eradicate tumors in select cases, resulting in significantly extended survival. Overall, this study not only unveils pivotal insights into the cytokines absent in PDAC that are crucial for promoting anti-tumor immunity but also pioneers an innovative immunotherapy approach.

Human T-cell leukemia virus type-1 (HTLV-1) is an oncogenic human retrovirus affecting over 20 million people worldwide. HTLV-1 infection can cause adult T-cell lymphoma (ATL) and other serious inflammatory diseases. Estimates report that 5-10% of HTLV-1 infected patients will develop a serious condition such as ATL, which has poor 4-year survival and high relapse rates. HTLV-1 has persistent infection rates across the globe and reaches up to 45% prevalence in certain communities. Despite this impact on human health, there are no direct-acting antivirals (DAAs) or vaccines against HTLV-1. HIV-1 and HTLV-1 are from the same viral family and encode for a homodimeric aspartyl protease crucial for cleavage of functional proteins from viral polyproteins. The activity of HIV-1 and HTLV-1 protease is essential to their viral life cycles. The Schiffer laboratory has extensive experience with viral protease crystallography and inhibition, especially with viral proteases for HIV-1, HCV NS3/4A, and SARS-CoV-2 main protease. This expertise uniquely positions me to design, synthesize, and characterize potent, resistance-thwarting protease inhibitors against HTLV-1 protease. Resistance-preventing DAA design is essential because of the selective pressure applied during DAA treatment. An ideal and proven strategy for developing a highly potent and resistance-preventing viral protease inhibitor is to target the active site through rational design using the substrate envelope. The substrate envelope for HTLV-1 protease has not been characterized and we lack a detailed understanding of the protease substrate specificity. I hypothesize that by translating strategies from our design of HIV-1 protease inhibitors, namely characterizing HTLV-1 protease’s substrate specificity, I can design potent and resistance-preventing DAAs for HTLV-1 protease. Aim 1: Characterize the structural basis for HTLV-1 protease substrate specificity. HTLV-1 protease cleaves six substrates by recognizing cleavage sites between individual proteins of the viral polyprotein. I will investigate the molecular basis of this recognition underlying protease specificity by determining cocrystal structures of the protease with bound substrates. The conserved volume inhabited by the substrates will define the substrate envelope and inform inhibitor design for HTLV-1 protease. Aim 2: Rationally design, synthesize, and characterize inhibitors of HTLV-1 protease to optimize potency. HIV-1 and HTLV-1 proteases share an active site amino acid sequence identity of 45% and high structural similarity. Therefore, I will begin inhibitor design by testing a selection of our in-house HIV-1 protease inhibitors, which have already shown low (1 µM) to moderate (30 nM) potency against HTLV-1 protease. I will combine experimental inhibition assays with cocrystal structure analysis to identify lead compounds for inhibitor design. I will leverage substrate specificity of the protease by moving inhibitor design towards compounds that mimic the shape of substrates, leveraging the substrate envelope (Aim 1), and the interactions between protease and substrate. I aim to produce novel, highly potent (sub-nM) inhibitors that will be promising DAAs for further investigation against HTLV-1.

Huntington’s Disease – a devastating neurodegenerative condition – is caused by a defect in the Huntingtin gene, resulting in the production of alternative forms of Huntingtin mRNA and protein. This proposal will use small RNA drugs to reduce alternative forms of Huntingtin in mouse models of Huntington’s disease and determine the effect on disease features and outcomes. Findings from these studies will provide insight into the mechanisms underlying Huntington’s Disease to inform the development of future therapeutics.

Mitochondria are essential for cell health and survival. Understanding the quality control machinery that mitochondria employ to maintain a healthy network is critical for health and disease. Our lab recently showed the role that lipid transfer protein Vps13D plays a critical role in mitochondrial clearance by autophagy (mitophagy) in the Drosophila developing midgut. Vps13D has been implicated in human movement disorders, highlighting the importance of understanding how it controls this process. Importantly, we do not know what proteins Vps13D may be interacting with at the mitochondrial surface to facilitate mitophagy. I performed an RNAi screen against mitochondrial genes that were shown to physically interact with Vps13D in human cells. I discovered that Mtch, the fly homolog of MTCH2, phenocopies both mitochondrial and autophagic defects that Vps13D mutants display, including failure to clear mitochondria, autophagic cargoes like p62, and the autophagy protein Atg8a. I generated a null mutant for Mtch, which displays phenotypes similar to what is observed by Mtch knockdown with RNAi and Vps13D mutants. Importantly, Mtch mutant cells exhibit a robust decrease in Vps13D protein puncta. I plan to use this Mtch mutant to: (1) characterize the function of Mtch in mitophagy, (2) determine the relationship between Mtch and Vps13D in mitophagy, and (3) investigate the relationship between Mtch and known regulators of autophagy and mitophagy. These studies will advance the field by creating a better understanding of mitophagy, and will also provide a novel genetic pathway to study that could lead to targeted therapies to correct mitochondrial disorders.

RNAi-based drugs are emerging as a new class of therapeutic modalities that are transforming pharmaceutical development. The National Institute of Arthritis and Musculoskeletal and Skin Diseases has granted a K99/R00 Pathway to Independence Award to Dr. Qi Tang to support his career transition to an independent principal investigator position at a U.S. academic institution and to fund his research on developing a programmable siRNA-based therapeutic platform for gene silencing in the skin. Dr. Tang will receive integrated scientific and career training during his K99 phase at UMass Chan, and in R00 phase, he will work to establish his independent laboratory with a focus on designing and expanding the utility of siRNA therapeutics for treating inflammatory skin diseases that currently lack sufficient treatments or are undruggable by conventional modalities.

Down syndrome (DS) is caused by trisomy for human chromosome 21 and affects more than five million individuals worldwide. Autism spectrum disorder (ASD) is a common co-occurring condition in persons with DS. Because individuals with DS-ASD often present with deleterious behaviours, such as self-injurious behaviour, aggression, sleep disturbances, and feeding difficulties, identifying effective treatments for the behavioural sequelae of DS-ASD has the potential to improve quality of life for individuals with DS and their caregivers. However, recent research has focused on diagnosing DS-ASD and describing its behavioural profile. The underlying molecular mechanisms that contribute to DS-ASD risk remain unexplored. Other DS-associated phenotypes likely result from dysregulation of the Sonic hedgehog (Shh) signalling pathway, which is a critical developmental pathway. Because Shh signalling is required for the differentiation of serotonergic neurons and because the serotonergic system is implicated in the pathogenesis of ASD, we hypothesized that disruption of Shh could result in behavioural phenotypes relevant to ASD. To address this hypothesis, we will perturb Shh signalling in developing zebrafish and assess serotonergic neuron morphology, overall brain structure, brain activity, and behaviour. How trisomy 21 contributes to misregulation of Shh also merits further study. Based on a previous screen of chromosome 21 genes, we selected HMGN1 as a likely regulator of Shh. We will overexpress HMGN1 and explore its effects on Shh signalling, serotonergic neurons, behaviour, gene expression, and epigenetic modifications. Successful completion of this project will lay the foundation for drug screens relevant to DS-ASD and cognition in larval zebrafish models.

Negative affect and stress experienced during alcohol abstinence can be a major factor contributing to relapse in alcohol use disorder, yet underlying neurobiological mechanisms remain ill-defined. Corticotropin-releasing factor (CRF)-expressing neurons in the bed nuclei of the stria terminalis (BNST) are involved in anxiety and stress responses, and they play a major role in the withdrawal. A subcommissural population of CRF neurons in the BNST (vBNSTCRF) is heavily innervated by hindbrain noradrenergic neurons that co-express prolactin releasing peptide (PrRP). Because these PrRP neurons are sensitive to both stress and interoceptive state, they are likely involved in the development of stress hypersensitivity following withdrawal. However, the role of PrRP neurons in alcohol-related behaviors has not been studied. I hypothesize that signaling of PrRP neurons to vBNST during acute ethanol withdrawal contributes to the development of negative affective behaviors, and that ethanol withdrawal potentiates vBNSTCRF responses to stress and PrRP neuronal activation. In the proposed project, mice will undergo chemogenetic silencing of PrRP+ neurons projecting to vBNST during acute ethanol withdrawal to examine the necessity of the circuit in the development of negative affective behaviors (Aim 1). Then, the influence of ethanol withdrawal on in vivo calcium responses of vBNSTCRF neurons to stress and chemogenetic activation of PrRP+ neurons will be explored using fiber photometry (Aim 2). Finally, monosynaptic tracing and whole brain imaging will be used to define additional brain regions innervating vBNSTCRF neurons that may be modulated concomitantly during ethanol withdrawal (Aim 3). The results of these studies will provide an improved understanding of neurobiological mechanisms impacting affective behavioral changes during ethanol abstinence. This project also provides a strong platform to expand and strengthen the trainee’s expertise in modern behavioral neuroscience approaches, including intersectional viral strategies for chemogenetic manipulation of neural circuits, observation of cell-type specific in vivo calcium signaling, and characterization of monosynaptic inputs to specific cell populations.

Regulation of innate immunological self-tolerance, or the ability of cells to discern “self” from “non-self” has long been studied in the periphery in autoimmune disorders, especially in the context of nucleic acids (NA). Understanding of self-tolerance in the central nervous system (CNS), however, has not been thoroughly investigated despite expression of these NA-sensing TLRs by microglia, the primary phagocyte of the CNS. Published data from our lab highlights that microglia retain untranslated RNA transcripts from engulfed myelin for days after phagocytosis in vitro and in human multiple sclerosis patients. Based on these data, I hypothesized that these retained transcripts could aberrantly activate endosomal TLRs. I, thus, induced primary demyelination in UNC93B1 -/- mice, which lack functional NA-sensing TLRs, and observed that these mice remyelinate more efficiently than wildtype. These data suggest that signaling of NA-sensing TLRs suppresses remyelination during demyelinating disease. Several exciting questions have now arisen, which I will tackle in this proposal: 1) Is myelin phagocytosis causing aberrant endosomal TLR signaling? 2) Are microglia the primary cell type driving this response? 3) Does a specific NA-sensing TLR hinder remyelination? I hypothesize that TLR7 is aberrantly signaling in response to engulfed myelin RNAs in microglia and suppressing remyelination. To address these questions, I have acquired powerful in vivo molecular genetic tools to manipulate UNC93B1 and endosomal TLR function. I will first identify molecular pathways that are changed in microglia in vitro in response to chronic myelin phagocytosis and test whether these molecules are UNC93B1-dependent (Aim 1a). I will then determine if the UNC93B1 dependent effects that I observed on remyelination are microglia-specific (Aim 1b). Lastly, I will identify the endosomal TLR underlying these UNC93B1 effects (Aim 2). I am now in a strong position to molecularly dissect the role of NA sensing TLRs in remyelination during demyelinating disease, which has high long-term therapeutic potential.

B cells are essential immune cells that protect the host from infections via antibody production. This requires B cells to acquire e9ector functions and di9erentiate from naïve into germinal centers, and eventually into antibody-secreting plasma cells. Cell- cell interactions underpinning e9ective B cell response have been extensively studied, yet, less focus has been placed on soluble factors involved in this process, notably, mechanistic insights into lipid production and sensing on B cell immunity is still lacking. To this end, I will characterize the role of the liver X-receptors (LXR), nuclear hormone receptors regulating cholesterol homeostasis, during a B cell response. I aim to understand, with the highest granularity, how B cells integrate intrinsic and extrinsic lipid metabolic cues. Using cutting edge approaches, including conditional and inducible murine knock-out models, dietary interventions, targeted epigenetic profiling, single-cell RNA sequencing (ScRNAseq), and spatial transcriptomic to 1) Investigate LXR requirements for germinal center B cell and plasma cell di9erentiation, proliferation and maintenance in both homeostasis, vaccination and infection, in di9erent tissues; 2) Elucidate the molecular mechanisms of LXR transcriptional activity and regulation in germinal centers and antibody-secreting plasma cells; and 3) Identify the natural LXR ligand(s) that specifically shape B cell responses. My research will help resolve with high granularity how lipid metabolite sensing tunes humoral immunity in steady state and inflammation. Furthermore, it will help better understand B cell immuno-metabolic circuits that are tuned by lipid metabolites and that might be leveraged to design harmacological interventions enhancing antibody-mediated immunity.

While there are many FDA-approved therapies to treat relapsing-remitting multiple Sclerosis (MS), there are far less options for treating neurodegeneration in progressive disease. Intriguingly, similar to other neurodegenerative diseases (e.g. Alzheimer’s disease and frontotemporal dementia), a hallmark feature of progressive disease in MS is the loss of synapses and gray matter atrophy. Our lab recently discovered a striking loss of synapses in the visual thalamus of MS patient tissue and MS-relevant mouse and marmoset models concomitant with visual impairment. This was particularly intriguing since prolonged visual impairment is historically attributed to demyelination of the optic nerve and is a frequent occurrence in MS patients. As complement proteins were previously shown to mediate synapse elimination by phagocytic microglia in neurodegenerative disease and genetic variants in complement proteins have recently been correlated with visual impairment in MS patients, Dr. Schafer’s lab has been exploring this pathway in synapse loss in the visual thalamus in MS. First, Dr. Schafer’s lab showed that complement proteins C1q and C3 were both increased in a mouse and non-human primate model of MS (experimental autoimmune encephalomyelitis, EAE). However, unlike in development, C1q did not localize to synapses in this context. Instead, C3 was highly synaptic in EAE to induce microglia-mediated phagocytosis and elimination of synaptic material. In contrast to C3 at synapses, Dr. Reich and Dr. Schafer identified that C1q was particularly high in microglia surrounding chronic active MS lesions and loss of C1q in microglia in the mouse EAE model attenuated the inflammatory response of microglia. Still, it is unclear how C1q is modulating inflammation, and whether C1q is working upstream of C3 to regulate synapse loss or if synapse loss is occurring through the alternative pathway, independent of C1q. Also, there are many other molecules that regulate complement proteins and it is unclear how many of these complement-related proteins contribute to MS-related disease. Therefore, the overall goal of this proposal is to gain a more comprehensive understanding of how complement proteins are regulated in demyelinating disease.